Animals and metal exposure
Roman snails (H. pomatia L.) were obtained from a commercial dealer (Exoterra, Dillingen, Germany). Garden snails (C. aspersus) were provided by the Department of Chrono-Environment, University of Franche-Comté, Besançon, France. All animals were reared under laboratory conditions (20°C, 80% humidity, 12:12 h photoperiod) at the Institute of Zoology in Innsbruck, Austria. Twenty-five snails from each species were split equally into five groups and fed over a period of 5 days on metal-enriched lettuce (Lactuca sativa). Metal loading of feed was achieved by soaking salad leaves in a corresponding metal salt solution (CdCl2 in H2O, with 1 and 3 mg Cd2+ L-1; ZnCl2 in H2O, with 5 and 10 mg Zn2+ L-1; CuCl2, with 10 mg Cu2+ L-1) . Resulting metal ion concentrations in the salad feed were as follows (means ± standard deviation, n = 5): Cd2+, 0.45 ± 0.11 and 1.12 ± 0.23 μmol g-1 dry weight; Cu2+, 3.51 ± 0.73 or 5.05 ± 0.97 μmol g-1 dry weight; Zn2+, 4.97 ± 3.42 and 6.93 ± 0.89 μmol g-1 dry weight). These concentrations range from physiologically to moderately elevated levels and are, therefore, representative for what could be the natural conditions encountered by snails. At days 0 and 5, RNA was extracted from the small midgut gland tissue aliquots (~10 mg fresh weight) of at least three animals and processed for cDNA synthesis as detailed below.
For in-situ-hybridization of HpCdMT isoform mRNAs, five individuals of H. pomatia were exposed over 14 days to a concentration of 14.97 μmol Cd g-1 dry weight. At the end of the exposure period, animals were sacrificed and their organs (midgut gland, midgut, kidney, mantle and foot) used for in-situ-hybridization analysis as described below.
Metal-enriched salad samples were oven-dried at 60°C for several days. Dried samples (snail tissues: 50-100 mg dry weight; salad samples: 100 - 500 mg dry weight) were wet-digested in screw-capped polypropylene tubes (Greiner, Kremsmünster, Austria) with a mixture of HNO3 (suprapure; Merck, Darmstadt, Germany) and distilled water (1:1) by heating at 70°C for several days. At the end of digestion, a few drops of H2O2 were added to the heated samples. The remaining solutions were diluted to a known volume with distilled water and analysed for metal concentrations (Cd, Zn, Cu) either by flame (model 2380 instrument, Perkin Elmer, Massachusetts, USA) or graphite furnace atomic absorption spectrophotometry (Hitachi Z-8200) with polarized Zeeman background correction (Hitachi, Tokyo, Japan).
Real-time detection PCR
RNA sampling for real-time detection PCR was done in control snails and animals exposed to metals over a 5 day period (see above). This time range was chosen because, in pulmonate snails, maximal induction of the CdMT gene by Cd2+ is reached only after several days. Total RNA was isolated from the homogenized midgut gland tissue of H. pomatia and C. aspersus individuals (Ultra Turrax T25, IKA Maschinenbau, Staufen, Germany) and quantified after DNaseI digestion (Fermentas, St Leon-Rot, Germany) by means of RiboGreen® RNA Quantitation Kit (Molecular Probes, OR, USA) with calibration curves derived from RNA standards using a fluorescence plate reader (Molecular Devices, CA, USA). Of total RNA, 450 ng was applied for cDNA synthesis using RevertAid™ H Minus M-MuLV Reverse Transcriptase (Fermentas) with hexamer primers in a 50 μL approach. Quantification of the RNA copy number was performed on a 7500 real-time PCR (RT-PCR) instrument from Applied Biosystems (CA, USA) using the Power SYBR Green approach (Applied Biosystems). Calibration curves from amplicon plasmids were used for copy number analysis for each of the MT isoforms involved, using primers designed with the Primer Express 3.0 software (Applied Biosystems) based on the known cDNA sequences for MT isoforms published in GenBank (H. pomatia HpCdMT and HpCuMT, accession numbers: AF399740 and AF399741, respectively; C. aspersus CaCdMT, CaCuMT and CaCd/CuMT, accession numbers: EF152281, EF178297, and EF206312). PCR primers used were as follows: HpCdMT: sense primer, 5'AAAGTGCACCTCAGCTTGCA 3'; antisense primer: 5' GCAGGCGGCACA TGTACAG 3'; amplicon length, 85 bp. HpCuMT: sense primer, 5' CCTTGCAGCTGTGGT AACGA 3'; antisense primer, 5' CAAGAACTGCATCGGTCACAA 3'; amplicon length, 65 bp; CaCdMT: sense primer, 5' GCCGCCTGTAAGACTTGCA 3'; antisense primer: 5' CACG CCTTGCCACACTTG 3'; amplicon length, 56 bp. CaCuMT: sense primer, 5' AACAGCAACCCTTGCAACTGT 3'; antisense primer, 5' CGAGCACTGCATTGATCACAA 3'; amplicon length, 74 bp. CaCd/CuMT: sense primer, 5' TGTGGAGCCGGCTGTTCT 3'; antisense primer, 5' CAGGTGTCATTGTTGCATTGG 3'; amplicon length, 59 bp. Optimal primer concentrations were determined by means of dissociation curves established for each primer pair. Two microlitres of cDNA were applied for RT detection PCR in a 20-μL approach (1x Power SYBR Green PCR Mastermix, 1x U-BSA, 900 mM sense primer, 300 mM antisense primer for HpCdMT and HpCuMT; 300 mM sense and 900 mM antisense primer for CaCdMT; 900 mM for sense and antisense primer for CaCuMT; 99 mM sense and 300 mM antisense primer for CaCd/CuMT). The PCR conditions were as follows: 50°C, 2 min; 95°C, 10 min; 40 repeats of 95°C, 15 s; and 60°C, 1 min.
In situ hybridization techniques
Cell- and tissue-specific expression of both HpMT isoforms was demonstrated by in situ hybridization (ISH). Construction of digoxigenin-11-UTP-labelled sense and antisense RNA probes for ISH of both MT isoform mRNAs, as well as ISH, antibody exposure and staining of parafomaldehyde-phosphate buffered saline (PBS)-fixed paraffin sections (5 μm) from tissues (midgut gland, midgut, kidney, mantle and foot) of control and metal-exposed animals were performed exactly as described previously . Control sections (exposed to either hybridization antisense or sense probes) were treated and incubated in the same way as samples but without anti-digoxigenin-alkaline phosphatase antibodies. For microscopy, all sections were embedded in Entellan (Merck) .
Construction of the recombinant expression vectors for wild-type Roman snail MT isoforms
The H. pomatia coding regions for both MT isoforms were amplified by PCR using the respective cDNAs synthesized during a previous investigation as a template . In order to facilitate their in frame cloning into the pGEX-4T1 expression vector (Amersham GE Healthcare Bio-Sciences AB, Uppsala, Sweden), BamHI and SalI restriction sites were generated just before the anti-thymocyte globulin (ATG) and after the stop codon. The oligonucleotides used for these PCR amplifications were: 5' ACAGGATCCGGACGAGGAAAGAACTGC 3' and 5' ATTGGATCCGGGAAAG GAAAAGGAGAAAAGTG 3' as HpCuMT and HpCdMT upstream primers, and 5' AGGCGTCGACTTGTCGTTTATTTGCAG 3' and 5' ATGCGTCGACTTGTCCTGC GGTTACT 3' as the HpCuMT and HpCdMT downstream primers. 35-cycle PCR reactions were performed under the following conditions: 94°C 30 s, 55°C 30 s and 72°C 30 s, using Deep Vent (New England Biolabs, Massachusetts, USA) thermostable DNA polymerase. PCR products were isolated from 2% agarose gels, digested with BamHI-SalI restriction enzymes (New England Biolabs) and cloned into the corresponding sites of pGEX-4T-1, for glutathione-S-transferase (GST)-MT fusion protein synthesis. The product resulting after purification was used in the second PCR as reverse megaprimer together with the forward primer mentioned earlier. In the final amplification product, the desired mutation had been introduced and the flanking restriction sites (BamHI and SalI) allowed the cloning in frame in pGEX-4T-1. Prior to the protein synthesis assays, all the DNA constructs were confirmed by automatic DNA sequencing (ABI 370, Perkin Elmer Life Sciences), using BigDye Terminator (Applied Biosystems). DH5α was the E. coli host strain used for cloning and sequencing purposes and, thereafter, the expression plasmids were transformed into the E. coli protease-deficient strain BL21 for recombinant protein overexpression.
Recombinant synthesis and purification of the metal-HpMT complexes
All HpMT metal complexes analysed in this work were biosynthesized in 2-L Erlenmeyer cultures of the corresponding transformed E. coli cells grown in LB medium with 100 mg mL-1 ampicillin and the following metal supplements: 300 μM ZnCl2 or CdCl2 for the zinc- or cadmium-rich media, or 500 μM CuSO4 for the copper-rich medium. Copper cultures were performed under two aeration conditions (high and low aeration) as previously described . GST-MT synthesis was induced with isopropyl-1-thio-β-D-galactopyranoside at a final concentration of 100 mM 30 min before the addition of the metal solution. After a 2.5 h-induction, cells were harvested by centrifugation. In order to prevent oxidation of the metal-HpMT complexes, argon was bubbled in all the steps of the purification following cell disruption.
For protein purification, cells were re-suspended in ice-cold PBS (1.4 M NaCl, 27 mM KCl, 101 mM Na2HPO4, 18 mM KH2PO4)-0.5% v/v β-mercaptoethanol, disrupted by sonication and centrifuged at 12,000 g for 30 min. The recovered supernatant was used to purify the GST-HpMT polypeptides by batch affinity chromatography with glutathione sepharose 4B (GE Healthcare, Buckinghamshire, UK) incubating the mixture with gentle agitation for 60 min at room temperature. After three washes in PBS and, since the GST-HpMT fusions include a thrombin recognition site, this protease was added (10 μ per mg of fusion protein) and digestion was carried out overnight at 23°-25°C. This allowed separation of the GST fragment of the fusion proteins, which remained bound to the gel matrix from the metal-HpMT portions that were eluted together with thrombin. Therefore, the eluate was concentrated using Centriprep Concentrators (Amicon; Millipore, MA, USA) with a cut-off of 3 kDa and subsequently fractionated using fast protein liquid chromatography (FPLC), through a Superdex-75 column (GE Healthcare) equilibrated with 50 mM Tris-HCl, pH 7.0, and run at 1 mL min-1. Fractions were collected and analysed for protein content by their absorbance at 254 nm. Aliquots of the protein-containing FPLC fractions were analysed by 15% SDS-PAGE and stained by Coomassie Blue. HpMT-containing samples were pooled and stored at -70°C until further use. Due to the specific recombinant expression conditions, the three synthesized snail MT isoforms contained one additional amino acid residue (G) at their N-termini in relation to the native isoforms previously isolated . These modifications do not interfere with the metal-binding capacity, as previously shown for both vertebrate  and invertebrate  MT isoforms.
Analysis of recombinantly expressed and in vitro prepared metal-HpMT complexes
The recombinantly expressed metal-MT complexes were analysed for element composition (S, Zn, Cd and Cu) by inductively coupled plasma atomic emission spectroscopy (ICP-AES) on a Polyscan 61E spectrometer (Thermo Jarrell Ash Corporation, MA, USA) at appropriate wavelengths (S, 182.040 nm; Zn, 213.856 nm; Cd, 228.802 nm; Cu, 324.803 nm), either under 'conventional' (dilution with 2% HNO3 (v/v)) or under 'acidic' (incubation in 1 M HCl at 65°C for 5 min) conditions . MT concentration in the recombinant preparations was calculated from the acidic ICP sulphur measurements, thus assuming the only contribution to their S content was that made by the HpCuMT and HpCdMT peptides. Protein concentrations were confirmed by standard amino acid analysis performed on an Alpha Plus Amino acid Autoanalyzer (Pharmacia LKB Biotechnology, Uppsala, Sweden) after sample hydrolysis in 6 M HCl (22 h at 110°C). Ser, Lys and Gly contents were used to extrapolate sample concentrations.
CD spectroscopy was performed using a model J-715 spectropolarimeter (JASCO, Gross-Umstadt, Germany) equipped with a computer (J-700 software, JASCO). Measurements were carried out at a constant temperature of 25°C maintained by a Peltier PTC-351 S apparatus (TE Technology Inc, MI, USA). Electronic absorption was measured on an HP-8453 diode-array ultra violet (UV)-vis spectrophotometer (GMI Inc, MN, USA), using 1-cm capped quartz cuvettes, and correcting for the dilution effects by means of the GRAMS 32 software (Thermo Fisher Scientific Inc, MA, USA).
Molecular mass determination was performed by electrospray ionization mass spectrometry equipped with a time-of-flight analyser (ESI-TOF MS) using a Micro Tof-Q Instrument (Brucker Daltonics GmbH, Bremen, Germany) calibrated with NaI (200 ppm NaI in a 1:1 H2O: isopropanol mixture), interfaced with a Series 1100 HPLC pump (Agilent Technologies, CA, USA) equipped with an autosampler, both controlled by the Compass Software. The experimental conditions for analysing proteins with divalent metals (Zn, Cd) were: 20 μL of the sample were injected through a PEEK long tube (1.5 m × 0.18 mm i.d.) at 40 μL/min under the following conditions: capillary-counterelectrode voltage, 5.0 kV; desolvation temperature, 90-110°C; dry gas 6 L/min. Spectra were collected throughout an m/z range from 800 to 2000. The proteins that contain copper were analysed injecting 20 μL of the sample at 30 μL/min; capillary-counterelectrode voltage, 4.0 kV; desolvation temperature, 80°C; m/z range from 800 to 2000. The liquid carrier was a 90:10 mixture of 15 mM ammonium acetate and acetonitrile, pH 7.0. For the analysis at acidic pH the conditions used were the same as those used in the analysis of the case for divalent metals, except in the composition of the carrier liquid which, in this case, was a 95:5 mixture of formic acid and acetonitrile at pH 2.4. All samples were injected at least in duplicate to ensure reproducibility. In all cases, molecular masses were calculated according to the reported method .
Metal replacement titrations were performed by adding the corresponding metal ions (Cd2+ or Cu+) at equivalent molar ratios to the recombinant Zn-HpMT complexes. Titrations were carried out following previously described procedures [64, 65]. The resulting in vitro complexes were analysed by UV-Vis and CD spectroscopy as well as mass spectrometry. All assays were carried out in an Ar atmosphere and the pH for all experiments remained constant throughout, without the addition of any extra buffers.
Metal tolerance complementation assays in transformed yeast MT-knockout cells
The Saccharomyces cerevisiae DTY4 strain (MATα, leu2-3, 112his3
1, trp1-1, ura3-50, gal1,
cup1::URA3) was used for metal tolerance complementation assays. This strain is characterized by a total MT deficiency due to cup1 disruption and Crs5 truncation .
The cDNAs coding for the different MTs assayed - the two snail MT isoforms (snail HpCdMT and snail HpCuMT), the two yeast MTs (Cup1 and Crs5) and the mouse MT1 - were ligated into the BamHI/PstI sites of the yeast vector p424, which contains TRP1 for selection, the constitutive GPD (glyceraldehyde-3-phosphate dehydrogenase) promoter for heterologous gene expression, and the CYC1 (cytochrome-c-oxidase) transcriptional terminator . The recombinant p424 vectors were introduced into the DTY4 cells using the lithium acetate procedure . Transformed cells were selected according to their capacity to grow in synthetic complete medium (SC) without Trp and Ura.
For metal tolerance tests, transformed yeast cells were initially grown in selective SC-Trp-Ura medium at 30°C and 220 rpm until saturation. These cells were then diluted to OD600 0.01 and used to re-inoculate tubes with 3 mL of fresh medium supplemented with CuSO4 added at 15, 30, 45, 60, 75, 90 and 105 μM final concentrations or CdCl2 at 1, 2, 4, 6 and 8 μM final concentrations. These cultures were allowed to grow for 18 h and the final OD600 was recorded and plotted as a percentage of the OD600 reached by the culture grown without metal supplement. For each concentration, and each kind of transformation, two replicas were run.
MT sequence alignment and phylogenetic analyses
MT amino acid sequences used for pulmonate MT alignments were derived mostly from amino acid sequence files or translated cDNA open reading frame sequences published in GenBank (http://www.ncbi.nlm.nih.gov/Tools/; see accession numbers in the legend to Figure 9). The editing and alignment were done manually in combination with ClustalX software Version 2.0.9 . For phylogenetic analyses, nucleotide sequences of the coding region of mollusc MT cDNAs or MT genes as well as protein primary sequences were used as published in GenBank (for accession numbers see legend of Figure 9). Phylogenetic reconstructions we performed with neighbour-joining  using the computer program PAUP* (version 4). The robustness of the phylogenetic hypothesis was tested by bootstrapping (1000 replicates) .